Field of the Invention
This invention relates to the genes encoding enzymes and regulatory proteins involved in dirhamnose-lipid biosynthesis. This invention also relates to a recombinant, nonpathogenic Pseudomonas chlororaphis which is capable of producing R1L and R2L.
The Sequence Listing submitted in text format (.txt) filed on Nov. 20, 2015, named “SequenceListing.txt”, (created on Nov. 19, 2014, 20.2 KB), is incorporated herein by reference.
Description of Related Art
Rhamnolipids are a family of rhamnose-containing glycolipids produced mainly by bacteria in the Pseudomonadaceae family, especially those belonging to the Pseudomonas genus. The lipid portion of most rhamnolipids contain 3-hydroxyalkanoyl-3-hydroxyalkanoate (Cx-Cy, where x and y are the carbon chain lengths of the alkanoate) moiety, though some rhamnolipids may contain only a monomeric 3-hydroxyalkanoate. Furthermore, rhamnolipid could also be synthesized with either one (R1L) or two (R2L) rhamnose molecules. Abdel-Mawgoud at al., 2010, Applied Microbiology and Biotechnology, 86:1323-1336, contains a summary of rhamnolipid varieties synthesized by various organisms. The structure of an R2L, i.e., α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate (R2-C10-C10), as an illustration, follows:

Rhamnolipids have many potential uses, most of which are associated with rhamnolipid's excellent surface-active properties. See, e.g., Faivre and Rosilio 2010, Expert Opinion on Drug Delivery, 7:1031-1048; Lourith and Kanlayavattanakul 2009; Nguyen and Sabatini 2011, International Journal at Cosmetic Science, 31:255-261; and Pinzon et al. 2009 In Hayes et al. (ed.). Biobased Surfactants and Detergents: Synthesis, Properties, and Applications, Chapter 4, pp. 77-105. AOCS Press, Urbana, Ill. Rhamnolipids may also possess valuable biological activities useful in wound healing (see Stipcevic, et al. 2006. Burns 32:24-34; see also U.S. Pat. No. 7,262,171), antibacterial (Sotirova, et al. 2008. Curr. Microbial. 56:639-644; Vatsa, et al. 2010. Int. J. Mol. Sci. 11:5095-5108), and fungicidal (Takemoto, et al. 2010. Am. J. Enol. Vitic. 61:120-124; Yoo, et al. 2005. J. Microbial. Biotechnol. 15:1164-1169) applications.
A putative metabolic pathway of rhamnolipid biosynthesis in P. aeruginosa is shown in FIG. 1. The precursor pool for rhamnolipid synthesis is proposed to be the fatty acid de novo biosynthesis pathway that could provide 3-ketoacyl-acyl carrier-protein (-ACP) metabolites with varying chain length of the acyl group. A β-ketoacyl reductase enzyme (RhlG) coded by the rhlG gene reduces the keto functional group into a hydroxyl group (Campos-García, et al. 1998. J. of Bacteriology, 180:4442-4451; Miller, et al. 2006. J. of Biological Chemistry 281:18025-18032). Two molecules of 3-hydroxyacyl-ACP are then condensed to yield 3-hydroxyalkanoyl-3-hydroxyalkanoate (3-HHA) by the rhamnosyltransferase A enzyme (RhlA) (Déziel, et al. 2003. Microbiology 149:2005-2013; Zhu and Rock 2008. J. of Bacteriology 190:3147-3154]. A rhamnose moiety from an activated sugar precursor, dTDP-rhamnose, is attached to 3-HHA to form R1L via the enzymatic action of rhamnosyltransferase B (RhlB) (Cabrera-Valladares, et al. 2006. Appl. Microbial. Biotechnol. 73:187-194). Finally, biosynthesis of R2L is accomplished by the transfer of another rhamnose moiety from dTDP-rhamnose to R1L by the action of rhamnosyltransferase C (RhlC) (Cabrera-Valladares, et al. 2006). Biochemical details of certain reaction steps in the pathway are still unclear. For example, it is not clear whether the active form of 3-HHA is attached to an ACP or not. Furthermore, the chemical-energy requirement in terms of high bond-energy molecules (e.g., ATP or dTTP) is not understood, leaving unanswered the question of how the dTDP-rhamnose molecule used as a substrate by RhlB and RhlC is regenerated.
Nevertheless, the overall picture of the metabolic pathway has allowed the genetic manipulation of bacteria to affect rhamnolipid synthesis. An early study demonstrated that several rhamnolipid-nonproducing bacteria (i.e. P. aeruginosa PG201, P. fluorescens ATC 15453, P. oleovorans GPol, P. putida KT2442, Escherichia coli DH5α, and E. coli W2190) could be genetically engineered to produce rhamnolipids through the expression of heterologous rhlA and rhlB genes from P. aeruginosa (Ochsner, et al. 1995. Appl. Environ. Microbiol. 61:3503-3506: Cabrera-Valladares, et al. 2006). Wang, et al. integrated P. aeruginosa rhlA and rhlB genes into the chromosome of E. coli BL21(DE3) and P. aeruginosa PAO1-rhlA− (Wang, et al. 2007. Biotechnology and Bioengineering 98:842-853). While the rhlA-rhlB-complemented P. aeruginosa transformant synthesized the same rhamnolipid mixture as that found in the wild-type P. aeruginosa, the E. coli transformant produced predominantly rhamnolipids having C10-C10 (ca. 60%) as the lipid component yields. Cha, et al. expressed rhlABRI gene cluster from P. aeruginosa EMS1 in P. putida 1067 to show production of rhamnolipids without detailing the compositions of the products (Cha, et al. 2008. Bioresource Technology 99:2192-2199).
P. aeruginosa is the most commonly studied organism for rhamnolipid biosynthesis. Various high-yield strains of P. aeruginosa have been adopted for large-scale production (Müller, et al. 2011. Applied Microbiology and Biotechnology 89:585-592). In view of rhamnolipid's potential applications in food (see U.S. Pat. No. 5,658,793) and medical (Stipcevic, et al. 2006; see U.S. Pat. No. 7,262,171) areas, a method for producing R1L using Pseudomonas chlororaphis, a nonpathogenic bacterium was previously developed (Gunther, et al. 2005. Appl. Environ. Microbiol. 71: 2288-2293; U.S. Pat. No. 7,202,063]. Despite this progress, there is still a need to develop a method for R2L in nonpathogenic bacterium. This invention identifies several genes involved in rhamnolipid biosynthesis and covers recombinant nonpathogenic bacteria capable of producing R1L in a 10-fold improved yield (in comparison to the original P. chlororaphis strain described in U.S. Pat. No. 7,202,063) or producing R2L, as a result of introduction of the appropriate DNA into the bacterium, thereby significantly lowering the production cost of R1L and broadening the application sphere of rhamnolipids.